Plants and seeds of corn variety LH249

ABSTRACT

According to the invention, there is provided seed and plants of the corn variety designated LH249. The invention thus relates to the plants, seeds and tissue cultures of the variety LH249, and to methods for producing a corn plant produced by crossing a corn plant of variety LH249 with itself or with another corn plant, such as a plant of another variety. The invention further relates to corn seeds and plants produced by crossing plants of variety LH249 with plants of another variety, such as another inbred line. The invention further relates to the inbred and hybrid genetic complements of plants of variety LH249.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to corn seed and plants of thevariety designated LH249, and derivatives and tissue cultures thereof.

2. Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop inbred plants frombreeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant of thevariety designated LH249. Also provided are corn plants having all thephysiological and morphological characteristics of the inbred cornvariety LH249. The inbred corn plant of the invention may furthercomprise, or have, a cytoplasmic or nuclear factor that is capable ofconferring male sterility or otherwise preventing self-pollination, suchas by self-incompatibility. Parts of the corn plant of the presentinvention are also provided, for example, pollen obtained from an inbredplant and an ovule of the inbred plant.

The invention also concerns seed of the inbred corn variety LH249. Theinbred corn seed of the invention may be provided as an essentiallyhomogeneous population of inbred corn seed of the variety designatedLH249. Essentially homogeneous populations of inbred seed are generallyfree from substantial numbers of other seed. Therefore, in the practiceof the present invention, inbred seed generally forms at least about 97%of the total seed. The population of inbred corn seed of the inventionmay be particularly defined as being essentially free from hybrid seed.The inbred seed population may be separately grown to provide anessentially homogeneous population of inbred corn plants designatedLH249.

In another aspect of the invention, a conversion of the corn varietyLH249 is provided. The conversion may comprise a genetic locus that is adominant or recessive allele. In certain embodiments of the invention,the genetic locus confers one or more traits such as, for example, malesterility, yield stability, waxy starch, yield enhancement, industrialusage, herbicide resistance, insect resistance, resistance to bacterial,fungal, nematode or viral disease, male fertility, and enhancednutritional quality. The genetic locus may be a naturally occurringmaize gene introduced into the genome of the variety by backcrossing, anatural or induced mutation, or a transgene introduced through genetictransformation techniques. When introduced through transformation, agenetic locus may comprise one or more transgenes integrated at a singlechromosomal location.

In yet another aspect of the invention, an inbred corn plant of thevariety designated LH249 is provided, wherein acytoplasmically-inherited trait has been introduced into said inbredplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continue to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of variety LH249 is provided. The tissue culture willpreferably be capable of regenerating plants capable of expressing allof the physiological and morphological characteristics of the variety,and of regenerating plants having substantially the same genotype asother plants of the variety. Examples of some of the physiological andmorphological characteristics of the variety LH249 includecharacteristics related to yield, maturity, and kernel quality, each ofwhich is specifically disclosed herein. The regenerable cells in suchtissue cultures will preferably be derived from embryos, meristematiccells, immature tassels, microspores, pollen, leaves, anthers, roots,root tips, silk, flowers, kernels, ears, cobs, husks, or stalks, or fromcallus or protoplasts derived from those tissues. Still further, thepresent invention provides corn plants regenerated from the tissuecultures of the invention, the plants having all the physiological andmorphological characteristics of variety LH249.

In yet another aspect of the invention, processes are provided forproducing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is aplant of the variety designated LH249. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second corn plant ofa different, distinct variety to provide a hybrid that has, as one ofits parents, the inbred corn plant variety LH249. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting, preferably in pollinating proximity, seeds of afirst and second parent corn plant, and preferably, seeds of a firstinbred corn plant and a second, distinct inbred corn plant. Where theplants are not in pollinating proximity, pollination can nevertheless beaccomplished by transferring a pollen or tassel bag from one plant tothe other as described below.

A second step comprises cultivating or growing the seeds of said firstand second parent corn plants into plants that bear flowers (corn bearsboth male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant). A third step comprisespreventing self-pollination of the plants, i.e., preventing the silks ofa plant from being fertilized by any plant of the same variety,including the same plant. This is preferably done by emasculating themale flowers of the first or second parent corn plant, (i.e., treatingor manipulating the flowers so as to prevent pollen production, in orderto produce an emasculated parent corn plant). Self-incompatibilitysystems may also be used in some hybrid crops for the same purpose.Self-incompatible plants still shed viable pollen and can pollinateplants of other varieties but are incapable of pollinating themselves orother plants of the same variety.

A fourth step comprises allowing cross-pollination to occur between thefirst and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is a plant of the variety designated LH249. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation (F₁) hybrid corn seed and plants producedby crossing an inbred in accordance with the invention with another,distinct inbred. The present invention further contemplates seed of anF₁ hybrid corn plant. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid corn plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe corn plant variety designated LH249 is provided. The phrase “geneticcomplement” is used to refer to the aggregate of nucleotide sequences,the expression of which sequences defines the phenotype of, in thepresent case, a corn plant, or a cell or tissue of that plant. A geneticcomplement thus represents the genetic make up of an inbred cell, tissueor plant, and a hybrid genetic complement represents the genetic make upof a hybrid cell, tissue or plant. The invention thus provides cornplant cells that have a genetic complement in accordance with the inbredcorn plant cells disclosed herein, and plants, seeds and diploid plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that variety LH249 and plants derived therefrom can beidentified by many types of genetic markers such as, for example, SimpleSequence Repeat polymorphisms (SSRs) (Williams et al., 1990), isozymes,Randomly Amplified Polymorphic DNAs (RAPDs), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified FragmentLength Polymorphisms (AFLPs) (EP 534 858, specifically incorporatedherein by reference in its entirety), and Single NucleotidePolymorphisms (SNPs) (Wang et al., 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the present invention provides a method ofproducing an inbred corn plant derived from the corn variety LH249, themethod comprising the steps of: (a) preparing a progeny plant derivedfrom corn variety LH249, wherein said preparing comprises crossing aplant of the corn variety LH249 with a second corn plant; (b) crossingthe progeny plant with itself or a second plant to produce a seed of aprogeny plant of a subsequent generation; (c) growing a progeny plant ofa subsequent generation from said seed of a progeny plant of asubsequent generation and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and (d) repeating steps (c)and (d) for an addition 3–10 generations to produce an inbred corn plantderived from the corn variety LH249. In the method, it may be desirableto select particular plants resulting from step (c) for continuedcrossing according to steps (b) and (c). By selecting plants having oneor more desirable traits, an inbred corn plant derived from the cornvariety LH249 is obtained which possesses some of the desirable traitsof corn variety LH249 as well potentially other selected traits.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions of Plant Characteristics

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. Rating times 10 is approximatelyequal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating: numerical ratings are based on aseverity scale where 1=most resistant to 9=susceptible.

Cn: Corynebacterium nebraskense rating. Rating times 10 is approximatelyequal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. Rating times 10 is approximatelyequal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating (values are percent plantsgirdled and stalk lodged).

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root ratings (1=least affected to 9=severepruning).

Ear-Attitude: The attitude or position of the ear at harvest scored asupright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, scored as white, pink, red, orbrown.

Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

Ear-Diameter: The average diameter of the ear at its midpoint.

Ear-Dry Husk Color: The color of the husks at harvest scored as buff,red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf scored as short,medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks. Minimum value no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest scored astight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of internodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence scored as green-yellow, yellow, pink, red, or purple.

Ear-Taper (Shape): The taper or shape of the ear scored as conical,semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating (values approximate percent ear rotted).

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units which are calculated by the Barger Method,where the heat units for a 24-h period are calculated as GDUs=[(Maximumdaily temperature+Minimum daily temperature)/2]−50. The highest maximumdaily temperature used is 86° F. and the lowest minimum temperature usedis 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for an inbred line or hybrid to have approximately 50% of theplants shedding pollen as measured from time of planting. GDUs to shedis determined by summing the individual GDU daily values from plantingdate to the date of 50% pollen shed.

GDUs to Silk: The number of growing degree units for an inbred line orhybrid to have approximately 50% of the plants with silk emergence asmeasured from time of planting. GDUs to silk is determined by summingthe individual GDU daily values from planting date to the date of 50%silking.

Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. +=indicates the presence of Htchlorotic-lesion type resistance; −=indicates absence of Htchlorotic-lesion type resistance; and +/−=indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm scored as white, paleyellow, or yellow.

Kernel-Endosperm Type: The type of endosperm scored as normal, waxy, oropaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp scored as colorless,red-white crown, tan, bronze, brown, light red, cherry red, orvariegated.

Kernel-Row Direction: The direction of the kernel rows on the ear scoredas straight, slightly curved, spiral, or indistinct (scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, scored as white, pale yellow, yellow, orange, red, or brown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel scored as dent, flint, or intermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. Rating times 10 is approximately equal topercent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk scored as upright (0to 30 degrees), intermediate (30 to 60 degrees), or lax (60 to 90degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollinationscored as light green, medium green, dark green, or very dark green.

Leaf-Length: The average length of the primary ear leaf.

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination. Rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf.

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf measured at itswidest point.

LSS: Late season standability (values times 10 approximate percentplants lodged in disease evaluation plots).

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating (1=resistant to 9=susceptible).

On2: Ostrinia nubilalis 2nd brood rating (1=resistant to 9=susceptible).

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale, where 1 equals best. The score is taken when the averageentry in a trial is at the fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating is actual percent infection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear measured from the groundto the point of attachment of the ear shank of the top developed ear tothe stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity). It is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis where 1 equals best.

STR: Stalk rot rating (values represent severity rating of 1=25% ofinoculated internode rotted to 9=entire stalk rotted and collapsed).

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating; numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions).

Tassel-Anther Color: The color of the anthers at 50% pollen shed scoredas green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination scored asopen or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed scored as green,red, or purple.

Tassel-Length: The length of the tassel measured from the base of thebottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle,measured from the base of the flag leaf to the base of the bottom tasselbranch.

Tassel-Pollen Shed: A visual rating of pollen shed determined by tappingthe tassel and observing the pollen flow of approximately five plantsper entry. Rated on a 1 to 9 scale where 9=sterile, 1=most pollen.

Tassel-Spike Length: The length of the spike measured from the base ofthe top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. Other Definitions

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Converted (Conversion) Plant: Plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of an inbred arerecovered in addition to one or more new trait conferred by the geneticlocus transferred into the inbred via the backcrossing technique. By“new trait,” it is understood that the trait may or may not be naturallyoccurring in maize, but is added or modified with respect to thestarting inbred. A genetic locus may comprise one or more genes. In thecase of transgenes, one or more transgenes are commonly integrated intoa host genome at a given locus. Such transgenes may comprise selectablemarkers, enhancers, or other components of the transformation vectorused.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Electrophoresis: A process by which particles suspended in a fluid or agel matrix are moved under the action of an electrical field, andthereby separated according to their charge and molecular weight. Thismethod of separation is well known to those skilled in the art and istypically applied to separating various forms of enzymes and of DNAfragments produced by restriction endonucleases.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Isozymes: Detectable variants of an enzyme, the variants catalyzing thesame reaction(s) but differing from each other, e.g., in primarystructure and/or electrophoretic mobility. The differences betweenisozymes are under single gene, codominant control. Consequently,electrophoretic separation to produce band patterns can be equated todifferent alleles at the DNA level. Structural differences that do notalter charge cannot be detected by this method.

Isozyme typing profile: A profile of band patterns of isozymes separatedby electrophoresis that can be equated to different alleles at the DNAlevel.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

SSR profile: A profile of simple sequence repeats used as geneticmarkers and scored by gel electrophoresis following PCR™ amplificationusing flanking oligonucleotide primers.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic sequence which has been introduced into the nuclearor chloroplast genome of a maize plant by a genetic transformationtechnique.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

III. Inbred Corn Plant LH249

A. Origin and Breeding History

LH249 was developed from an initial cross of LH236×LH242 followed byselfing and use of the pedigree system of plant breeding. Yield, stalkquality, root quality, disease tolerance, late plant greenness, lateplant intactness, ear retention, pollen shedding ability, silkingability and corn borer tolerance were the criteria used to determine therows from which ears were selected.

LH236 and LH242 are both proprietary field corn inbred lines of Holden'sFoundation Seeds, LLC. PVPA certificate 9700003 was issued for LH236 onMay 29, 1998. U.S. Pat. No. 5,731,504, issued Mar. 4, 1998, alsoprotects LH236. PVPA certificate 9700076 was issued for LH242 on May 29,1998. U.S. Pat. No. 5,750,850, May 12, 1998, also protects LH242.

LH249 has shown uniformity and stability for all traits. It has beenself-pollinated and ear-rowed a sufficient number of generations, withcareful attention to uniformity of plant type to ensure homozygosity andphenotypic stability. The line has been increased both by hand (Iowa2001 and 2002; Hawaii 2002) and sibbed in isolated production fields(Hawaii 2003 and Iowa 2003) with continued observations for uniformity.The originating plant breeder, has observed LH249 all five generationsit has been increased. The line is stable, uniform and no variant traitshave been observed or are anticipated in LH249.

Inbred corn plants can be reproduced by planting the seeds of the inbredcorn plant LH249, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts. Seeds can be harvested from such a plant usingstandard, well known procedures.

B. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a corn plant having the physiological and morphologicalcharacteristics of corn plant LH249. A description of the physiologicaland morphological characteristics of corn plant LH249 is presented inTable 1.

TABLE 1 Physiological and Morphological Traits for the LH249 PhenotypeTYPE: Dent REGION WHERE DEVELOPED: Northcentral U.S. MATURITY: Days HeatUnits From emergence to 50% of plants in silk: 69 1386 From emergence to50% of plants in pollen 69 1386${Heat}{\mspace{11mu}\;}{Units}\text{:} = \frac{\begin{matrix}\left\lbrack {{{Max}.\mspace{14mu}{Temp}.\left( {\leq {86{^\circ}\mspace{20mu}{F.}}} \right)} +} \right. \\{\left. {{{{Min}.\mspace{14mu}{Temp}}..}\left( {\underset{\_}{>}{50{^\circ}\mspace{20mu}{F.}}} \right)} \right\rbrack - 50}\end{matrix}}{2}$ PLANT: Plant Height (to tassel tip): 222.7 cm EarHeight (to base of top ear): 86.8 cm Length of Top Ear Internode: 13.9cm Average Number of Ears per Stalk: 1.0 Anthocyanin of Brace Roots:Moderate LEAF: Width of Ear Node Leaf: 8.0 cm Length of Ear Node Leaf:70.3 cm Number of leaves above top ear: 6.2 Leaf Angle (from 2nd Leafabove ear at anthesis to Stalk above leaf): 22.0° Leaf Color: DarkGreen - Munsell Code 5 GY 3/4 Leaf Sheath Pubescence (Rate on scale from1 = none to 9 = like peach fuzz): 3 Marginal Waves (Rate on scale from 1= none to 9 = many): 5 Longitudinal Creases (Rate on scale from 1 = noneto 9 = many): 6 TASSEL: Number of Lateral Branches: 8.0 Branch Anglefrom Central Spike: 31.0° Tassel Length (from top leaf collar to tasseltop): 37.2 cm Anther Color: Yellow - Munsell Code 2.5 Y 8/10 GlumeColor: Purple - Munsell Code 5 RP 5/8 Bar Glumes: Absent EAR: (UnhuskedData) Silk Color (3 days after emergence): Tan - Munsell Code 2.5 GY 8/6with 5 R 5/8 Fresh Husk Color (25 days after 50% silking): Light Green -Munsell Code 2.5 GY 7/6 Dry Husk Color (65 days after 50% silking):Buff - Munsell Code 7.5 YR 7/4 Position of Ear: Upright Husk Tightness(Rate on scale from 1 = very loose to 9 = very tight): 9 Husk Extension:Short (ears exposed) EAR: (Husked Ear Data) Ear Length: 13.7 cm EarDiameter at mid-point: 41.6 mm Number of Kernel Rows: 14.8 Kernel Rows:Distinct Row Alignment: Straight Shank Length: 8.6 cm Ear Taper: AverageKERNEL: (Dried) Kernel Length: 10.8 mm Kernel Width: 7.6 mm KernelThickness: 4.4 mm Aleurone Color Pattern: Homozygous Aleurone Color:White - Munsell Code 2.5Y 8/2 Hard Endosperm Color: Yellow - MunsellCode 2.5Y 7/8 Endosperm Type: Normal Starch COB: Cob Diameter atMid-Point: 25.2 mm Cob Color: Pink - Munsell code 5 R 6/6 AGRONOMICTRAITS: 0% Dropped Ears (at 65 days after anthesis) 0% Pre-anthesisBrittle Snapping 0% Pre-anthesis Root Lodging 0% Post-anthesis RootLodging (at 65 days after anthesis) *These are typical values. Valuesmay vary due to environment. Other values that are substantiallyequivalent are also within the scope of the invention.

C. Deposit Information

A representative deposit of 2500 seeds of the inbred corn varietydesignated LH249 has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. on Nov. 7, 2006. Thosedeposited seeds have been assigned ATCC Accession No. PTA-7975. Thedeposit was made in accordance with the terms and provisions of theBudapest Treaty relating to deposit of microorganisms and was made for aterm of at least thirty (30) years and at least five (05) years afterthe most recent request for the furnishing of a sample of the deposit isreceived by the depository, or for the effective term of the patent,whichever is longer, and will be replaced if it becomes non-viableduring that period.

IV. Conversions of Variety LH249

When the term inbred corn plant is used in the context of the presentinvention, this also includes any conversions of that inbred. It isunderstood to those of skill in the art that, using backcrossing,essentially any locus may be introduced into the novel corn varietydescribed herein. The term converted plant as used herein refers tothose corn plants which are developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of an inbred arerecovered in addition to a genetic locus transferred into the inbred viathe backcrossing technique.

Backcrossing methods can be used with the present invention to improveor introduce a trait into the inbred. The term backcrossing as usedherein refers to the repeated crossing of a hybrid progeny back to oneof the parental corn plants for that inbred. The parental corn plantwhich contributes the locus or loci for the desired trait is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur.

The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). In a typical backcross protocol,the original inbred of interest (recurrent parent) is crossed to asecond inbred (nonrecurrent parent) that carries the genetic locus ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the transferredlocus from the nonrecurrent parent. The backcross process may beaccelerated by the use of genetic markers, such as SSR, RFLP, SNP orAFLP markers to identify plants with the greatest genetic complementfrom the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto add or substitute one or more new traits in the original inbred. Toaccomplish this, a genetic locus of the recurrent inbred is modified orsubstituted with the desired locus from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological constitution ofthe original inbred. The choice of the particular nonrecurrent parentwill depend on the purpose of the backcross; one of the major purposesis to add some commercially desirable, agronomically important trait tothe plant. The exact backcrossing protocol will depend on thecharacteristic or trait being altered to determine an appropriatetesting protocol. Although backcrossing methods are simplified when thecharacteristic being transferred is a dominant allele, a recessiveallele may also be transferred. In this instance it may be necessary tointroduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new inbred but that can be improved by backcrossingtechniques. A genetic locus conferring the traits may or may not betransgenic. Examples of such traits known to those of skill in the artinclude, but are not limited to, male sterility, waxy starch, herbicideresistance, resistance for bacterial, fungal, or viral disease, insectresistance, male fertility, enhanced nutritional quality, industrialusage, yield stability, and yield enhancement. These genes are generallyinherited through the nucleus, but may be inherited through thecytoplasm. Some known exceptions to this are genes for male sterility,some of which are inherited cytoplasmically, but still act as a singlelocus trait. A number of exemplary genetic loci conferring new traitsare described in, for example, PCT Application WO 95/06128, thedisclosure of which is specifically incorporated herein by reference.

Examples of genes conferring male sterility include those disclosed inU.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat. No.4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. A particularly useful type of malesterility gene is one which can be induced by exposure to a chemicalagent, for example, a herbicide (U.S. patent Ser. No. 08/927,368, filedSep. 11, 1997, the disclosure of which is specifically incorporatedherein by reference in its entirety). Both inducible and non-induciblemale sterility genes can increase the efficiency with which hybrids aremade, in that they eliminate the need to physically emasculate the cornplant used as a female in a given cross.

Where one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, where cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns the inbred corn plant LH249 comprising a genetic locus capableof restoring male fertility in an otherwise male-sterile inbred orhybrid plant. Examples of male-sterility genes and correspondingrestorers which could be employed with the inbred of the invention arewell known to those of skill in the art of plant breeding and aredisclosed in, for instance, U.S. Pat. No. 5,530,191; U.S. Pat. No.5,689,041; U.S. Pat. No. 5,741,684; and U.S. Pat. No. 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

Direct selection may be applied where a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofmaize are known to those of skill in the art. For example, methods whichhave been described for the genetic transformation of maize includeelectroporation (U.S. Pat. No. 5,384,253), electrotransformation (U.S.Pat. No. 5,371,003), microprojectile bombardment (U.S. Pat. No.5,550,318; U.S. Pat. No. 5,736,369, U.S. Pat. No. 5,538,880; and PCTPublication WO 95/06128), Agrobacterium-mediated transformation (U.S.Pat. No. 5,591,616 and E.P. Publication EP672752), direct DNA uptaketransformation of protoplasts (Omirulleh et al., 1993) and siliconcarbide fiber-mediated transformation (U.S. Pat. No. 5,302,532 and U.S.Pat. No. 5,464,765).

A type of trait which can be introduced by genetic transformation (U.S.Pat. No. 5,554,798) and has particular utility is a gene which confersresistance to the herbicide glyphosate. Glyphosate inhibits the actionof the enzyme EPSPS, which is active in the biosynthetic pathway ofaromatic amino acids. Inhibition of this enzyme leads to starvation forthe amino acids phenylalanine, tyrosine, and tryptophan and secondarymetabolites derived therefrom. Mutants of this enzyme are availablewhich are resistant to glyphosate. For example, U.S. Pat. No. 4,535,060describes the isolation of EPSPS mutations which confer glyphosateresistance upon organisms having the Salmonella typhimurium gene forEPSPS, aroA. A mutant EPSPS gene having similar mutations has also beencloned from Zea mays. The mutant gene encodes a protein with amino acidchanges at residues 102 and 106 (PCT Publication WO 97/04103). When aplant comprises such a gene, a herbicide resistant phenotype results.

Plants having inherited a transgene comprising a mutated EPSPS gene may,therefore, be directly treated with the herbicide glyphosate without theresult of significant damage to the plant. This phenotype providesfarmers with the benefit of controlling weed growth in a field of plantshaving the herbicide resistance trait by application of the broadspectrum herbicide glyphosate. For example, one could apply theherbicide ROUNDUP™, a commercial formulation of glyphosate manufacturedand sold by the Monsanto Company, over the top in fields whereglyphosate resistant corn plants are grown. The herbicide applicationrates may typically range from 4 ounces of ROUNDUP™ to 256 ouncesROUNDUP™ per acre. More preferably, about 16 ounces to about 64 ouncesper acre of ROUNDUP™ may be applied to the field. However, theapplication rate may be increased or decreased as needed, based on theabundance and/or type of weeds being treated. Additionally, depending onthe location of the field and weather conditions, which will influenceweed growth and the type of weed infestation, it may be desirable toconduct further glyphosate treatments. The second glyphosate applicationwill also typically comprise an application rate of about 16 ounces toabout 64 ounces of ROUNDUP™ per acre treated. Again, the treatment ratemay be adjusted based on field conditions. Such methods of applicationof herbicides to agricultural crops are well known in the art and aresummarized in general in Anderson (1983).

Alternatively, more than one trait may be introgressed into an eliteinbred by the method of backcross conversion. A selectable marker geneand a gene encoding a protein which confers a trait of interest may besimultaneously introduced into a maize plant as a result of genetictransformation. Usually one or more introduced genes will integrate intoa single chromosome site in the host cell's genome. For example, aselectable marker gene encoding phosphinothricin acetyl transferase(PPT) (e.g., a bar gene) and conferring resistance to the activeingredient in some herbicides by inhibiting glutamine synthetase, and agene encoding an endotoxin from Bacillus thuringiensis (Bt) andconferring resistance to particular classes of insects, e.g.,lepidopteran insects, in particular the European Corn Borer, may besimultaneously introduced into a host genome. Furthermore, through theprocess of backcross conversion more than one transgenic trait may betransferred into an elite inbred.

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

It is understood to those of skill in the art that a single locus oftransgenic origin need not be directly transformed into a plant, astechniques for the production of stably transformed corn plants thatpass single loci to progeny by Mendelian inheritance is well known inthe art. Such single loci may therefore be passed from parent plant toprogeny plants by standard plant breeding techniques that are well knownin the art.

Many thousands of single locus traits are known to those of skill in theart and may be transferred into the variety described herein using wellknown techniques. Non-limiting examples of some such single locus traitsthat may be introduced into a plant of the invention according to wellknown methods are as follows: the uidA gene from E. Coli encodingβ-glucuronidase (GUS) (cells expressing uidA produce a blue color whengiven the appropriate substrate, Jefferson, R. A. 1987. Plant Mol. Biol.Rep 5: 387–405); the bar gene from Streptomyces hygroscopicus encodingphosphinothricin acetyltransferase (PAT) (cells expressing PAT areresistant to the herbicide Basta, White et al., 1990. Nucl. Ac. Research18: 1062); the lux gene from firefly encoding luciferase (cellsexpressing lux emit light under appropriate assay conditions, deWet etal., 1987. Mol. Cell. Biol. 7: 725–737); the dhfr gene from mouseencoding dihydrofolate reductase (DHFR) (cells expressing dhfr areresistant to methotrexate; Eichholtz et al., 1987. Somatic Cell Mol.Genet. 13: 67–76); the neo gene from E. coli encoding aminoglycosidephosphotransferase (APH) (cells expressing neo are resistant to theaminoglycoside antibiotics; Beck et al., 1982. Gene 19: 327–336); theamp gene from E. Coli encoding β-lactamase (cells expressing β-lactamaseproduce a chromogenic compound when given the appropriate substrate;Sutcliffe, J. G. 1978. Proc. Nat. Acad. Sci. USA 75: 3737–3741); thexylE gene from Ps. putida encoding catechol dihydroxygenase (cellsexpressing xylE produce a chromogenic compound when given theappropriate substrate; Zukowsky et al., 1983. Proc. Nat. Acad. Sci. USA80: 1101–1105); the R, C1 and B genes from maize encode proteins thatregulate anthocyanin biosynthesis in maize (Goff et al., 1990. EMBOJ.:2517–2522); the ALS gene from Zea mays encoding acetolactate synthaseand mutated to confer resistance to sulfonylurea herbicides (cellsexpressing ALS are resistant to the herbicide; Gleen et al., 1992. PlantMolecular Biology 18: 1185–1187); the proteinase inhibitor II gene frompotato and tomato (plants expressing the proteinase inhibitor II geneshow increased resistance to insects; potato—Graham et al., 1986. Mol.Cell. Biol. 2: 1044–1051; tomato—Pearce et al., 1991. Science253:895–898); the Bt gene from Bacillus thuringiensis berliner 1715encoding a protein that is toxic to insects (this gene is the codingsequence of Bt 884 modified in two regions for improved expression inplants; Vaeck et al., 1987. Nature 328: 33–37); the bxn gene fromKlebsiella ozaeneae encoding a nitrilase enzyme specific for theherbicide bromoxynil (cells expressing this gene are resistant to theherbicide bromoxynil; Stalker et al., Science 242: 419–422, 1988); theWGA-A gene encoding wheat germ agglutinin (expression of the WGA-A geneconfers resistance to insects; Smith and Raikhel, 1989. Plant Mol.Biology 13: 601–603); the dapA gene from E. coli encodingdihydrodipicolinate synthase (expression of this gene in plant cellsproduces increased levels of free lysine; Richaud et al., 1986. J.Bacteriol. 166: 297–300); the Z10 gene encoding a 10 kD zein storageprotein from maize (expression of this gene in cells alters thequantities of 10 kD Zein in the cells; Kirihara et al., 1988. Mol. Gen.Genet. 211: 477–484); the Bt gene cloned from Bacillus thuringiensisKurstaki encoding a protein that is toxic to insects (the gene is thecoding sequence of the cry IA(c) gene modified for improved expressionin plants—plants expressing this gene are resistant to insects; Höfteand Whiteley, 1989. Microbiological Reviews. 53: 242–255); the ALS genefrom Arabidopsis thaliana encoding a sulfonylurea herbicide resistantacetolactate synthase enzyme (cells expressing this gene are resistantto the herbicide Gleen et al., 1988. Mol. Gen. Genet. 211: 266–271); thedeh1 gene from Pseudomonas putida encoding a dehalogenase enzyme (cellsexpressing this gene are resistant to the herbicide Dalapon;Buchanan-Wollaston et al., 1992. Plant Cell Reports 11: 627–631); thehygromycin phosphotransferase II gene from E. coli (expression of thisgene in cells produces resistance to the antibiotic hygromycin; Waldronet al., 1985. Plant Molecular Biology 5: 103–108); the mtlD gene clonedfrom E. coli (the gene encodes the enzyme mannitol-1-phosphatedehydrogenase; Lee and Saier, 1983. J. of Bacteriol. 153:685); the HVA-1gene encoding a Late Embryogenesis Abundant (LEA) protein (the gene wasisolated from barley; Dure et al., Plant Molecular Biology 12: 475–486);a short amino acid sequence able to specify nuclear location, (Steifelet al., 1984. Cell 39:499–509); a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation (PlantCell 2:785–793, 1990); plant disease resistance genes such as thoseactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen (Jones et al., 1994. Science266:7891), the tomato Cf-9 gene for resistance to Cladospotium (Martinet al., 1993. Science 262: 1432), a tomato Pto gene for resistance toPseudomonas syringae pv. (Lblindrinos et al., 1994. Cell 78: 1089),Arabidopsis RSP2 gene for resistance to Pseudomonas syringae; a geneconferring resistance to a pest, such as soybean cyst nematode (PCTApplication W096/30517; PCT Application W093/19181), a lectin (Van Dammeet al., 1994. Plant Molec. Biol. 24:25, a vitamin-binding protein suchas avidin (PCT application US93/06487); an enzyme inhibitor, forexample, a protease or proteinase inhibitor or an amylase inhibitor (Abeet al., 1987. J. Biol. Chem. 262:16793), a nucleotide sequence of ricecysteine proteinase inhibitor (Huub et al., 1993. Plant Molec. Biol.21:985), a nucleotide sequence of encoding tobacco proteinase inhibitorI (Sumitani et al., 1993. Biosci. Biotech. Biochem. 57:1243), anucleotide sequence of Streptomyces nitrosporeus a-amylase inhibitor(U.S. Pat. No. 5,494,813); an insect-specific hormone or pheromone suchas an ecysteroid and juvenile hormone, a variant thereof, a mimeticbased thereon, or an antagonist or agonist thereof (Hammock et al.,1990. Nature 344:458); an insect-specific peptide or neuropeptide which,upon expression, disrupts the physiology of the affected pest (Regan,1994. J. Biol. Chem. 269:9); an insect-specific venom produced in natureby a snake, a wasp, etc. (Pang et al., 1992. Gene 116:165), an enzymeinvolved in the modification, including the post-translationalmodification, of a biologically active molecule; for example, aglycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease,a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, akinase, a phosphorylase, a polymerase, an elastase, a chitinase and aglucanase, whether natural or synthetic (PCT application WO 93/02197),which discloses the nucleotide sequence of a callase gene; DNA moleculeswhich contain chitinase-encoding sequences (Kramer et al., 1993. InsectBiochem. Molec. Biol. 23:691), who teach the nucleotide sequence of acDNA encoding tobacco hookworm chitinase, and Kawalleck et al., 1993.Plant Molec. Biol. 21 :673), who provide the nucleotide sequence; amolecule that stimulates signal transduction (Botella et al., 1994.Plant Molec. Eiol. 24:757) of nucleotide sequences for mung beancalmodulin cDNA clones, and (Griess et al., 1994. Plant Physiol,104:1467), who provide the nucleotide sequence of a maize calmodulincDNA clone; a sequence of the parsley ubi4-2 polyubiquitin gene peptide(PCT application W095/16776); a membrane permease, a channel former or achannel blocker (Jaynes et al., 1993. Plant Sci 89:43), ofheterologous-expression of a cecropin-P, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum; aviral-invasive protein or a complex toxin derived therefrom, forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses (Beachy et al., 1990. Ann. rev. Phytopathol.28:451); an insect-specific antibody or an immunotoxin derived therefrom(Cf. Taylor et al., 1994. Abstract W97, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland); avirus-specific antibody (Tavladoraki et al., 1993. Nature 366:469), whoshow that transgenic plants expressing recombinant antibody genes areprotected from virus attack; a developmental-arrestive protein producedin nature by a pathogen or a parasite by solubilizing plant cell wallhomo-a-I, 4-D-galacturonase (Lamb et al., 1992. Biotechnology 10:1436);a gene encoding a bean endopolygalacturonase-inhibiting protein (Toubartet al., 1992. Plant J. 2:367); a development-arrestive protein producedin nature by a plant (Logemann et al., 1992. Biotechnology 10:305);genes that confer resistance to a herbicide, for example, a herbicidethat inhibits the growing point or meristem, such as a mutant ALS andAHAS enzyme (Lee et al., 1988. EMBO J. 7:1241; Miki et al., 1990. Theor.Appl. Genet. 80:449), and pyridinoxy or phenoxy proprionic acids andcycloshexones (ACCase inhibitor-encoding genes) (U.S. Pat. No.4,940,835, Shah, et al.), which discloses the nucleotide sequence of aform of EPSP which can confer glyphosate resistance, the nucleotidesequence of a phosphinothricin-acetyl-transferase gene in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al., 1989.Biotechnology 7:61; genes conferring resistance to phenoxy proprionicacids and clycloshexones, such as sethoxydim and haloxyfop, includingAccl-S1, Accl-S2 and Accl-S3 genes (Marshall et al., 1992. Theor. Appl.Genet. 83:4:35); a herbicide that inhibits photosynthesis, such as atriazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene)(Przibila et al., 1991. Plant Cell 3:169); mutant psbA genes; nitrilasegenes (U.S. Pat. No. 4,810,648 to Stalker); modified fatty acidmetabolism genes, for example, an antisense gene of stearoyl-ACPdesaturase to increase stearic acid content of the plant (Knutzon etal., 1992. Proc. Natl. Acad. Sci. U.S.A. 89:2624); a phytase-encodinggene to enhance breakdown of phytate, adding more free phosphate to thetransformed plant (Van Hartingsveldt et al., 1993. Gene 127:87); anAspergillus niger phytase gene; a gene coding for an enzyme that altersthe branching pattern of starch; also (Shiroza et al., 1988. J. Bact.170:810) (nucleotide sequence of Streptococcus mutansfructosyltransferase gene), (Steinmetz et al., 1985. Mol. Gen. Genet.20:220) (nucleotide sequence of Bacillus subtilis levansucrase gene),(Pen et al., 1992. Biotechnology 10:292) (production of transgenicplants that express Bacillus lichenifonnis mamylase), (Elliot et al.,1993. Plant Molec. Biol. 21 515) (nucleotide sequences of tomatoinvertase genes), Sergaard et al., 1993. J. Biol. Chem. 268:22480)(site-directed mutagenesis of barley a-amylase gene), and (Fisher etal., 1993. Plant Physiol. 102: 1 045) (maize endosperm starch branchingenzyme II).

A. Origin and Breeding History of an Exemplary Converted Plant

85DGD1 MLms is a conversion of 85DGD1 to cytoplasmic male sterility.85DGD1 MLms was derived using backcross methods. 85DGD1 (a proprietaryinbred of Monsanto Company) was used as the recurrent parent and MLms, agermplasm source carrying ML cytoplasmic sterility, was used as thenonrecurrent parent. The breeding history of the converted inbred 85DGD1MLms can be summarized as follows:

Hawaii Nurseries Planting Made up S-O: Female row 585 male row 500 DateApr. 2, 1992 Hawaii Nurseries Planting S-O was grown and plants werebackcrossed Date Jul. 15, 1992 times 85DGD1 (rows 444 ′ 443) HawaiiNurseries Planting Bulked seed of the BC1 was grown and Date Nov. 18,1992 backcrossed times 85DGD1 (rows V3-27 ′ V3-26) Hawaii NurseriesPlanting Bulked seed of the BC2 was grown and Date Apr. 2, 1993backcrossed times 85DGD1 (rows 37 ′ 36) Hawaii Nurseries Planting Bulkedseed of the BC3 was grown and Date Jul. 14, 1993 backcrossed times85DGD1 (rows 99 ′ 98) Hawaii Nurseries Planting Bulked seed of BC4 wasgrown and Date Oct. 28, 1993 backcrossed times 85DGD1 (rows KS-63 ′KS-62) Summer 1994 A single ear of the BC5 was grown and backcrossedtimes 85DGD1 (MC94-822 ′ MC94-822-7) Winter 1994 Bulked seed of the BC6was grown and backcrossed times 85DGD1 (3Q-1 ′ 3Q-2) Summer 1995 Seed ofthe BC7 was bulked and named 85DGD1 MLms.

B. Illustrative Procedures for Production of Converted Plants

As described above, techniques for the production of converted cornplants are well known in the art (see, e.g., Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). A non-limiting example of such aprocedure one of skill in the art would use for preparation of aconversion of corn plant LH249 is as follows:

-   -   (a) crossing corn plant LH249 to a second (nonrecurrent) corn        plant comprising a locus to be converted in corn plant LH249;    -   (b) selecting at least a first progeny plant resulting from the        crossing and comprising the locus;    -   (c) crossing the selected progeny to corn plant LH249; and    -   (d) repeating steps (b) and (c) until a plant of variety LH249        is obtained comprising the locus.

Following these steps, essentially any locus may be introduced into cornplant LH249. The locus can be introduced without regard to whether thelocus confers any given trait. For example, molecular techniques allowintroduction of any given locus, without the need for phenotypicscreening of progeny during the backcrossing steps.

PCR and Southern hybridization are two examples of molecular techniquesthat may be used for confirmation of the presence of a given locus andthus conversion of that locus. The techniques are carried out asfollows: Seeds of progeny plants are grown and DNA isolated from leaftissue (see Sambrook et al., 1989; Shure et al. 1983). Approximately onegram of leaf tissue is lyophilized overnight in 15 ml polypropylenetubes. Freeze-dried tissue is ground to a power in the tube using aglass rod. Powdered tissue is mixed thoroughly with 3 ml extractionbuffer (7.0 urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1%sarcosine). Tissue/buffer homogenate is extracted with 3 mlphenol/chloroform. The aqueous phase is separated by centrifugation, andprecipitated twice using 1/10 volume of 4.4 M ammonium acetate pH 5.2,and an equal volume of isopropanol. The precipitate is washed with 75%ethanol and resuspended in 100–500 μl TE (0.01 M Tris-HCI, 0.001 M EDTA,pH 8.0). The DNA may then be screened as desired for presence of thelocus.

For PCR, two hundred to 1000 ng genomic DNA from the progeny plant beingscreened is added to a reaction mix containing 10 mM Tris-HCl pH 8.3,1.5 mM MgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP,dGTP, dTTP, 20% glycerol, 2.5 units Taq DNA polymerase and 0.5 μM eachof forward and reverse DNA primers that span a segment of the locusbeing converted. The reaction is run in a thermal cycling machine 3minutes at 94 C, 39 repeats of the cycle 1 minute at 94 C, 1 minute at50 C, 30 seconds at 72 C, followed by 5 minutes at 72 C. Twenty μl ofeach reaction mix is run on a 3.5% NuSieve gel in TBE buffer (90 mMTris-borate, 2 mM EDTA) at 50V for two to four hours. The amplifiedfragment is detected using an agarose gel. Detection of an amplifiedfragment corresponding to the segment of the locus spanned by theprimers indicates the presence of the locus.

For Southern analysis, plant DNA is restricted, separated in an agarosegel and transferred to a Nylon filter in 10×SCP (20 SCP: 2M NaCl, 0.6 Mdisodium phosphate, 0.02 M disodium EDTA) according to standard methods(Southern, 1975). Locus DNA or RNA sequences are labeled, for example,radioactively with ³²P by random priming (Feinberg & Vogelstein, 1983).Filters are prehybridized in 6×SCP, 10% dextran sulfate, 2% sarcosine,and 500 μg/ml denatured salmon sperm DNA. The labeled probe isdenatured, hybridized to the filter and washed in 2×SCP, 1% SDS at 65°for 30 minutes and visualized by autoradiography using Kodak XAR5 film.Presence of the locus is indicated by detection of restriction fragmentsof the appropriate size.

VI. Tissue Cultures and in Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated LH249. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

One type of tissue culture is tassel/anther culture. Tassels containanthers which in turn enclose microspores. Microspores develop intopollen. For anther/microspore culture, if tassels are the plantcomposition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., 1987), trypan blue (preferred) and acetocarmine squashing. Themid-uninucleate microspore stage has been found to be the developmentalstage most responsive to the subsequent methods disclosed to ultimatelyproduce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3 M mannitol combined with 50mg/l of ascorbic acid, silver nitrate, and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

In embodiments where microspores are obtained from anthers, microsporescan be released from the anthers into an isolation medium following themannitol preculture step. One method of release is by disruption of theanthers, for example, by chopping the anthers into pieces with a sharpinstrument, such as a razor blade, scalpel, or Waring blender. Theresulting mixture of released microspores, anther fragments, andisolation medium are then passed through a filter to separatemicrospores from anther wall fragments. An embodiment of a filter is amesh, more specifically, a nylon mesh of about 112 mm pore size. Thefiltrate which results from filtering the microspore-containing solutionis preferably relatively free of anther fragments, cell walls, and otherdebris.

Isolation of microspores can be accomplished at a temperature belowabout 25° C. and preferably, at a temperature of less than about 15° C.Preferably, the isolation media, dispersing tool (e.g., razor blade),funnels, centrifuge tubes, and dispersing container (e.g., petri dish)are all maintained at the reduced temperature during isolation. The useof a precooled dispersing tool to isolate maize microspores has beenreported (Gaillard et al., 1991).

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6% sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin, and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. An illustrativeembodiment of a solid support is a TRANSWELL® culture dish. Anotherembodiment of a solid support for development of the microspores is abilayer plate wherein liquid media is on top of a solid base. Otherembodiments include a mesh or a millipore filter. Preferably, a solidsupport is a nylon mesh in the shape of a raft. A raft is defined as anapproximately circular support material which is capable of floatingslightly above the bottom of a tissue culture vessel, for example, apetri dish, of about a 60 or 100 mm size, although any other laboratorytissue culture vessel will suffice. In an illustrative embodiment, araft is about 55 mm in diameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent).

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertswhich support the microspores at the surface of 2 ml of medium.

After the microspores have been isolated, they can be cultured in a lowstrength anther culture medium until about the 50 cell stage when theyare subcultured onto an embryoid/callus maturation medium. Medium isdefined at this stage as any combination of nutrients that permit themicrospores to develop into embryoids or callus. Many examples ofsuitable embryoid/callus promoting media are well known to those skilledin the art. These media will typically comprise mineral salts, a carbonsource, vitamins, and growth regulators. A solidifying agent isoptional. A preferred embodiment of such a media is referred to as “Dmedium,” which typically includes 6N1 salts, AgNO₃ and sucrose ormaltose.

In an illustrative embodiment, 1 ml of isolated microspores are pipettedonto a 10 mm nylon raft and the raft is floated on 1.2 ml of medium “D,”containing sucrose or preferably maltose. Both calli and embryoids candevelop. Calli are undifferentiated aggregates of cells. Type I is arelatively compact, organized, and slow growing callus. Type II is asoft, friable, and fast-growing one. Embryoids are aggregates exhibitingsome embryo-like structures. The embryoids are preferred for subsequentsteps to regenerating plants. Culture medium “D” is an embodiment ofmedium that follows the isolation medium and replaces it. Medium “D”promotes growth to an embryoid/callus. This medium comprises 6N1 saltsat ⅛ the strength of a basic stock solution (major components) and minorcomponents, plus 12% sucrose, or preferably 12% maltose, 0.1 mg/l B1,0.5 mg/l nicotinic acid, 400 mg/l proline and 0.5 mg/l silver nitrate.Silver nitrate is believed to act as an inhibitor to the action ofethylene. Multi-cellular structures of approximately 50 cells eachgenerally arise during a period of 12 days to 3 weeks. Serial transferafter a two week incubation period is preferred.

After the petri dish has been incubated for an appropriate period oftime, preferably two weeks in the dark at a predefined temperature, araft bearing the dividing microspores is transferred serially to solidbased media which promote embryo maturation. In an illustrativeembodiment, the incubation temperature is 30° C. and the mesh raftsupporting the embryoids is transferred to a 100 mm petri dishcontaining the 6N1-TGR-4P medium, an “anther culture medium.” Thismedium contains 6N1 salts, supplemented with 0.1 mg/l TIBA, 12% sugar(sucrose, maltose, or a combination thereof), 0.5% activated charcoal,400 mg/l proline, 0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2 percentGELRITE™ (solidifying agent) and is capable of promoting the maturationof the embryoids. Higher quality embryoids, that is, embryoids whichexhibit more organized development, such as better shoot meristemformation without precocious germination, were typically obtained withthe transfer to full strength medium compared to those resulting fromcontinuous culture using only, for example, the isolated microsporeculture (IMC) Medium “D.” The maturation process permits the pollenembryoids to develop further in route toward the eventual regenerationof plants. Serial transfer occurs to full strength solidified 6N1 mediumusing either the nylon raft, the TRANSWELL® membrane, or bilayer plates,each one requiring the movement of developing embryoids to permitfurther development into physiologically more mature structures. In anespecially preferred embodiment, microspores are isolated in anisolation media comprising about 6% maltose, cultured for about twoweeks in an embryoid/calli induction medium comprising about 12% maltoseand then transferred to a solid medium comprising about 12% sucrose.

At the point of transfer of the raft, after about two weeks ofincubation, embryoids exist on a nylon support. The purpose oftransferring the raft with the embryoids to a solidified medium afterthe incubation is to facilitate embryo maturation. Mature embryoids atthis point are selected by visual inspection indicated by zygoticembryo-like dimensions and structures and are transferred to the shootinitiation medium. It is preferred that shoots develop before roots, orthat shoots and roots develop concurrently. If roots develop beforeshoots, plant regeneration can be impaired. To produce solidified media,the bottom of a petri dish of approximately 100 mm is covered with about30 ml of 0.2% GELRITE™ solidified medium. A sequence of regenerationmedia are used for whole plant formation from the embryoids.

During the regeneration process, individual embryoids are induced toform plantlets. The number of different media in the sequence can varydepending on the specific protocol used. Finally, a rooting medium isused as a prelude to transplanting to soil. When plantlets reach aheight of about 5 cm, they are then transferred to pots for furthergrowth into flowering plants in a greenhouse by methods well known tothose skilled in the art.

Plants have been produced from isolated microspore cultures by themethods disclosed herein, including self-pollinated plants. The rate ofembryoid induction was much higher with the synergistic preculturetreatment consisting of a combination of stress factors, including acarbon source which can be capable of inducing starvation, a coldtemperature, and colchicine, than has previously been reported. Anillustrative embodiment of the synergistic combination of treatmentsleading to the dramatically improved response rate compared to priormethods, is a temperature of about 10° C., mannitol as a carbon source,and 0.05% colchicine.

The inclusion of ascorbic acid, an anti-oxidant, in the isolation mediumis preferred for maintaining good microspore viability. However, thereseems to be no advantage to including mineral salts in the isolationmedium. The osmotic potential of the isolation medium was maintainedoptimally with about 6% sucrose, although a range of 2% to 12% is withinthe scope of this invention.

In an embodiment of the embryoid/callus organizing media, mineral saltsconcentration in IMC Culture Media “D” is (⅛×), the concentration whichis used also in anther culture medium. The 6N1 salts major componentshave been modified to remove ammonium nitrogen. Osmotic potential in theculture medium is maintained with about 12% sucrose and about 400 mg/lproline. Silver nitrate (0.5 mg/l) was included in the medium to modifyethylene activity. The preculture media is further characterized byhaving a pH of about 5.7 to 6.0. Silver nitrate and vitamins do notappear to be crucial to this medium but do improve the efficiency of theresponse.

Examples of processes of tissue culturing and regeneration of corn aredescribed in, for example, European Patent Application 0 160 390, Greenand Rhodes (1982) and Duncan et al. (1985), Songstad et al. (1988), Raoet al. (1986), Conger et al. (1987), PCT Application WO 95/06128,Armstrong and Green, 1985; Gordon-Kamm et al., 1990, and U.S. Pat. No.5,736,369.

VII. Processes of Preparing Corn Plants and the Corn Plants Produced bySuch Crosses

The present invention provides processes of preparing novel corn plantsand corn plants produced by such processes. In accordance with such aprocess, a first parent corn plant is crossed with a second parent cornplant wherein at least one of the first and second corn plants is theinbred corn plant LH249. One application of the process is in theproduction of F₁ hybrid plants. Another important aspect of this processis that it can be used for the development of novel inbred lines. Forexample, the inbred corn plant LH249 could be crossed to any secondplant, and the resulting hybrid progeny each selfed for about 5 to 7 ormore generations, thereby providing a large number of distinct,pure-breeding inbred lines. These inbred lines could then be crossedwith other inbred or non-inbred lines and the resulting hybrid progenyanalyzed for beneficial characteristics. In this way, novel inbred linesconferring desirable characteristics could be identified.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand. In one embodiment, crossing comprises the steps of:

-   -   (a) planting in pollinating proximity seeds of a first and a        second parent corn plant, and preferably, seeds of a first        inbred corn plant and a second, distinct inbred corn plant;    -   (b) cultivating or growing the seeds of the first and second        parent corn plants into plants that bear flowers;    -   (c) emasculating flowers of either the first or second parent        corn plant, i.e., treating the flowers so as to prevent pollen        production, or alternatively, using as the female parent a male        sterile plant, thereby providing an emasculated parent corn        plant;    -   (d) allowing natural cross-pollination to occur between the        first and second parent corn plants;    -   (e) harvesting seeds produced on the emasculated parent corn        plant; and, where desired,    -   (f) growing the harvested seed into a corn plant, preferably, a        hybrid corn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant LH249 is employed asthe male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factorwhich causes resistance to the emasculating effects of the chemicalagent. Gametocides affect processes or cells involved in thedevelopment, maturation or release of pollen. Plants treated with suchgametocides are rendered male sterile, but typically remain femalefertile. The use of chemical gametocides is described, for example, inU.S. Pat. No. 4,936,904, the disclosure of which is specificallyincorporated herein by reference in its entirety. Furthermore, the useof Roundup herbicide in combination with glyphosate tolerant maizeplants to produce male sterile corn plants is disclosed in U.S. patentapplication Ser. No. 08/927,368 and PCT Publication WO 98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

Both parental inbred plants of corn may be allowed to continue to growuntil maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

Alternatively, in another embodiment of the invention, both first andsecond parent corn plants can be from variety LH249. Thus, any cornplant produced using corn plant LH249 forms a part of the invention. Asused herein, crossing can mean selfing, backcrossing, crossing toanother or the same inbred, crossing to populations, and the like. Allcorn plants produced using the inbred corn plant LH249 as a parent are,therefore, within the scope of this invention.

A. F₁ Hybrid Corn Plant and Seed Production

One beneficial use of the instant corn variety is in the production ofhybrid seed. Any time the inbred corn plant LH249 is crossed withanother, different, corn inbred, a first generation (F₁) corn hybridplant is produced. As such, an F₁ hybrid corn plant can be produced bycrossing LH249 with any second inbred maize plant. Essentially any othercorn plant can be used to produce a hybrid corn plant having corn plantLH249 as one parent. All that is required is that the second plant befertile, which corn plants naturally are, and that the plant is not cornvariety LH249.

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. Typically, F₁ hybrids are more vigorous than their inbredparents. This hybrid vigor, or heterosis, is manifested in manypolygenic traits, including markedly improved yields, better stalks,better roots, better uniformity and better insect and diseaseresistance. In the development of hybrids only the F₁ hybrid plants aretypically sought. An F₁ single cross hybrid is produced when two inbredplants are crossed. A double cross hybrid is produced from four inbredplants crossed in pairs (A×B and C×D) and then the two F₁ hybrids arecrossed again (A×B)×(C×D).

Thousands of corn varieties are known to those of skill in the art, anyone of which could be crossed with corn plant LH249 to produce a hybridplant. For example, the U.S. Patent & Trademark has issued more than 300utility patents for corn varieties. The Maize Genetics Cooperation StockCenter, which is supported by the U.S. Department of Agriculture, has atotal collection approaching 80,000 individually pedigreed samples(//w3.ag.uiuc.edu/maize-coop/mgc-info.html).

Therefore, any F₁ hybrid corn plant or corn seed which is produced withLH249 as a parent is part of the present invention. Examples of such F₁hybrid which has been produced with LH249 as a parent are given in Table2. When the inbred corn plant LH249 is crossed with another inbred plantto yield a hybrid, it can serve as either the maternal or paternalplant.

B. Development of Corn Varieties Using LH249

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing corn variety LH249 followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing corn variety LH249 with any secondplant. In selecting such a second plant to cross for the purpose ofdeveloping novel inbred lines, it may be desired to choose those plantswhich either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Examples of potentially desired characteristicsinclude greater yield, better stalks, better roots, resistance toinsecticides, herbicides, pests, and disease, tolerance to heat anddrought, reduced time to crop maturity, better agronomic quality, highernutritional value, and uniformity in germination times, standestablishment, growth rate, maturity, and fruit size.

Once initial crosses have been made with corn variety LH249, inbreedingtakes place to produce new inbred varieties. Inbreeding requiresmanipulation by human breeders. Even in the extremely unlikely eventinbreeding rather than crossbreeding occurred in natural corn,achievement of complete inbreeding cannot be expected in nature due towell known deleterious effects of homozygosity and the large number ofgenerations the plant would have to breed in isolation. The reason forthe breeder to create inbred plants is to have a known reservoir ofgenes whose gametic transmission is predictable.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desired characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed and selected insuccessive generations. Each succeeding generation becomes morehomogeneous as a result of self-pollination and selection. Typically,this method of breeding involves five or more generations of selfing andselection: S₁→S₂; S₂→S₃; S₃→S₄; S₄→S₅, etc. After at least fivegenerations, the inbred plant is considered genetically pure.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. Field trials are used over several yearsfor the progressive elimination of hybrids based on detailed evaluationof their phenotype. Eventually, strip trials are conducted to formallycompare the experimental hybrids being developed with other hybrids,some of which were previously developed and generally are commerciallysuccessful. That is, comparisons of experimental hybrids are made tocompetitive hybrids to determine if there was any advantage to furtherdevelopment of the experimental hybrids. Examples of such comparisonsare presented hereinbelow. After testing is complete, determinations maybe made whether commercial development should proceed for a givenhybrid.

C. F₁ Hybrid Comparisons

As mentioned above, hybrids are progressively eliminated followingdetailed evaluations of their phenotype, including formal comparisonswith other commercially successful hybrids. Strip trials are used tocompare the phenotypes of hybrids grown in as many environments aspossible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

Examples of such comparative data are set forth hereinbelow in Table 2,which presents a comparison of performance data for a hybrid made withLH249 as one parent, versus selected hybrids of commercial value.

TABLE 2 Comparative Data for a Hybrid Having LH249 as One Inbred ParentMean Plant Height Ear Height Test Pedigree Yield % M Y/M % Stalk % Root% Drop (cm) (cm) Weight LH249 X LH273 193 17.4 11.04 3 2 0 114 44 52.6LH236 X LH273 1 −2.6 1.49 −1 −1 0 3 3 −2.3 LH312BT1 X LH273 −11 −1.20.13 −1 −1 0 2 −2 −1.7 LH245 X LH279 −9 −0.7 −0.06 −2 0 0 9 2 −1.9 LH249X LH287 192 20.2 9.48 4 2 0 116 40 54.9 LH310 X LH262 8 −2.7 1.47 1 2 0−7 −9 −1.6 LH311 X LH287 4 −1.5 0.86 0 −4 0 −3 −1 0.0 LH310 X LH287 9−1.0 0.88 0 −2 0 0 0 0.7 LH249 X LH322 193 18.4 10.53 2 0 0 102 37 55.5LH310 X LH172 −2 −1.1 0.53 0 0 0 −3 −3 0.5 LH246 X LH287 −26 −1.1 −0.73−1 0 0 −9 −2 −1.8 LH310 X LH322 −2 −0.8 0.34 0 0 0 −2 −1 −0.8VIII. Genetic Complements

The present invention provides a genetic complement of the inbred cornplant variety designated LH249. Further provided by the invention is ahybrid genetic complement, wherein the complement is formed by thecombination of a haploid genetic complement from LH249 and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of acorn plant or a cell or tissue of that plant. By way of example, a cornplant is genotyped to determine a representative sample of the inheritedmarkers it possesses. Markers are alleles at a single locus. They arepreferably inherited in codominant fashion so that the presence of bothalleles at a diploid locus is readily detectable, and they are free ofenvironmental variation, i.e., their heritability is 1. This genotypingis preferably performed on at least one generation of the descendantplant for which the numerical value of the quantitative trait or traitsof interest are also determined. The array of single locus genotypes isexpressed as a profile of marker alleles, two at each locus. The markerallelic composition of each locus can be either homozygous orheterozygous. Homozygosity is a condition where both alleles at a locusare characterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

A genetic marker profile of an inbred may be predictive of the agronomictraits of a hybrid produced using that inbred. For example, if an inbredof known genetic marker profile and phenotype is crossed with a secondinbred of known genetic marker profile and phenotype it is possible topredict the phenotype of the F₁ hybrid based on the combined geneticmarker profiles of the parent inbreds. Methods for prediction of hybridperformance from genetic marker data is disclosed in U.S. Pat. No.5,492,547, the disclosure of which is specifically incorporated hereinby reference in its entirety. Such predictions may be made using anysuitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

SSRs are genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can behighly informative in linkage analysis relative to other marker systemsin that multiple alleles may be present. Another advantage of this typeof marker is that, through use of flanking primers, detection of SSRscan be achieved, for example, by the polymerase chain reaction (PCR™),thereby eliminating the need for labor-intensive Southern hybridization.The PCR™ detection is done by use of two oligonucleotide primersflanking the polymorphic segment of repetitive DNA. Repeated cycles ofheat denaturation of the DNA followed by annealing of the primers totheir complementary sequences at low temperatures, and extension of theannealed primers with DNA polymerase, comprise the major part of themethodology. Following amplification, markers can be scored by gelelectrophoresis of the amplification products. Scoring of markergenotype is based on the size (number of base pairs) of the amplifiedsegment.

Means for performing genetic analyses using SSR polymorphisms are wellknown in the art. The SSR analyses reported herein were conducted byCelera AgGen in Davis, Calif. This service is available to the public ona contractual basis. This analysis was carried out by amplification ofsimple repeats followed by detection of marker genotypes using gelelectrophoresis. Markers were scored based on the size of the amplifiedfragment.

The SSR genetic marker profile of the parental inbreds and exemplaryresultant hybrid described herein were determined. Because an inbred isessentially homozygous at all relevant loci, an inbred should, in almostall cases, have only one allele at each locus. In contrast, a diploidgenetic marker profile of a hybrid should be the sum of those parents,e.g., if one inbred parent had the allele 168 (base pairs) at aparticular locus, and the other inbred parent had 172, the hybrid is168.172 by inference. Subsequent generations of progeny produced byselection and breeding are expected to be of genotype 168, 172, or168.172 for that locus position. When the F₁ plant is used to produce aninbred, the locus should be either 168 or 172 for that position.Surprisingly, it has been observed that in certain instances, novel SSRgenotypes arise during the breeding process.

Another aspect of this invention is a plant genetic complementcharacterized by a genetic isozyme typing profile. Isozymes are forms ofproteins that are distinguishable, for example, on starch gelelectrophoresis, usually by charge and/or molecular weight. Thetechniques and nomenclature for isozyme analysis are described in, forexample, Stuber et al. (1988), which is incorporated by reference.

A standard set of loci can be used as a reference set. Comparativeanalysis of these loci is used to compare the purity of hybrid seeds, toassess the increased variability in hybrids compared to inbreds, and todetermine the identity of seeds, plants, and plant parts. In thisrespect, an isozyme reference set can be used to develop genotypic“fingerprints.”

The present invention also provides a hybrid genetic complement formedby the combination of a haploid genetic complement of the corn plantLH249 with a haploid genetic complement of a second corn plant. Meansfor combining a haploid genetic complement from the foregoing inbredwith another haploid genetic complement can comprise any method forproducing a hybrid plant from LH249. It is contemplated that such ahybrid genetic complement can be prepared using in vitro regeneration ofa tissue culture of a hybrid plant of this invention.

A hybrid genetic complement contained in the seed of a hybrid derivedfrom LH249 is a further aspect of this invention. The geneticcomplements of a hybrid may be assessed by any of the many well knowntechniques as described above.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A seed of corn variety LH249, wherein a sample of the seed of thecorn variety LH249 was deposited under ATCC Accession No. PTA-7975.
 2. Acorn plant of corn variety LH249, wherein a sample of the seed of thecorn variety LH249 was deposited under ATCC Accession No. PTA-7975.
 3. Aplant part of the corn plant of claim
 2. 4. The plant part of claim 3,further defined as pollen, an ovule or a cell.
 5. A corn plantexpressing all of the physiological and morphological characteristics ofthe corn plant of claim
 2. 6. The corn plant of claim 2, furthercomprising a nuclear or cytoplasmic gene conferring male sterility.
 7. Amethod of producing a male sterile corn plant comprising introducing anucleic acid molecule that confers male sterility into the plant ofclaim
 2. 8. A male sterile corn plant produced by the method of claim 6.9. A tissue culture of cells of a plant of corn variety LH249, wherein asample of the seed of the corn variety LH249 was deposited under ATCCAccession No. PTA-7975.
 10. The tissue culture of claim 9, wherein thecells are obtained from embryos, immature embryos, meristematic cells,immature tassels, microspores, pollen, leaves, anthers, roots, roottips, silk, flowers, kernels, ears, cobs, husks, or stalks.
 11. A cornplant regenerated from the tissue culture of claim 9, wherein the cornplant expresses all of the physiological and morphologicalcharacteristics of corn variety LH249, wherein a sample of the seed ofthe corn variety LH249 was deposited under ATCC Accession No. PTA-7975.12. A process of producing corn seed, comprising crossing a first parentcorn plant with a second parent corn plant, wherein one or both of thefirst parent corn plant or the second parent corn plant is a plant ofcorn variety LH249, wherein a sample of the seed of the corn varietyLH249 was deposited under ATCC Accession No. PTA-7975, wherein seed isallowed to form.
 13. The process of claim 12, further defined as aprocess of producing hybrid corn seed, comprising crossing a plant ofcorn variety LH249 with a second, distinct corn plant, wherein a sampleof the seed of the corn variety LH249 was deposited under ATCC AccessionNo. PTA-7975.
 14. The process of claim 13, wherein crossing comprisesthe steps of: (a) planting the seeds of first and second inbred cornplants; (b) cultivating the seeds of said first and second inbred cornplants into plants that bear flowers; (c) preventing self pollination ofat least one of the first or the second inbred corn plant; (d) allowingcross-pollination to occur between the first and second inbred cornplants; and (e) harvesting seeds on at least one of the first or secondinbred corn plants, said seeds resulting from said cross-pollination.15. The corn plant of claim 2, further comprising a transgene introducedby genetic transformation.
 16. The corn plant of claim 15, wherein thetransgene confers a trait selected from the group consisting ofherbicide tolerance, insect resistance, disease resistance, waxy starch,decreased phytate content, modified fatty acid metabolism, modifiedcarbohydrate metabolism, male sterility and restoration of malefertility.
 17. A method of producing a transgenic corn plant, comprisingintroducing a transgene into a plant of corn variety LH249, wherein asample of the seed of the corn variety LH249 was deposited under ATCCAccession No. PTA-7975.
 18. A method of producing an inbred corn plantderived from the corn variety LH249, the method comprising the steps of:(a) preparing a progeny plant derived from corn variety LH249 bycrossing a plant of the corn variety LH249 with a second corn plant,wherein a sample of the seed of the corn variety LH249 was depositedunder ATCC Accession No. PTA-7975; (b) crossing the progeny plant withitself or a second plant to produce a seed of a progeny plant of asubsequent generation; (c) growing a progeny plant of a subsequentgeneration from said seed and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and (d) repeating steps (b)and (c) for an additional 2–10 generations to produce an inbred cornplant further derived from the corn variety LH249.
 19. A method ofproducing a conversion of the corn variety LH249 to express at least onenew trait, the method comprising the steps of: (a) crossing a first cornplant having a first diploid genome comprising a plurality of pairedchromosomes comprising a plurality of mappable genetic loci with a pairof alleles at each locus, and further comprising a genetic locus thatconfers at least one new trait, with a second plant of the corn varietyLH249, a sample of the seed of the corn variety LH249 having beendeposited under ATCC Accession No. PTA-7975, the plant of the cornvariety LH249 having a second diploid genome comprising a plurality ofpaired chromosomes comprising a plurality of mappable genetic loci witha pair of alleles at each locus, to produce seed comprising a diploidgenome having a plurality of paired chromosomes comprising a pluralityof mappable genetic loci with a pair of alleles at each locus, whereinone of the alleles is contributed by the first corn plant and the otheris contributed by the plant of the corn variety LH249, said genomefurther comprising the genetic locus that confers the new trait; (b)harvesting and planting the seed thereby produced to produce at leastone progeny plant of the first filial generation, said progeny plantcomprising a diploid genome comprising the genetic locus; (c) crossingsaid progeny plant with a plant of the corn variety LH249 to produceseed of a subsequent filial generation, the seed comprising a diploidgenome having a plurality of paired chromosomes comprising a pluralityof mappable genetic loci with a pair of alleles at each locus, whereinone of the alleles is contributed by the progeny plant and the other iscontributed by the plant of the corn variety LH249, and furthercomprising the genetic locus that confers the new trait; (d) growing atleast one progeny plant of the subsequent filial generation from theseed produced in step (c), said progeny plant comprising a genomecomprising the genetic locus that confers the new trait; (e) repeatingsteps (c) and (d) for at least three additional generations to produce aconverted plant of the corn variety LH249 wherein the plant comprises adiploid genome having a plurality of paired chromosomes comprising aplurality of mappable genetic loci with a pair of alleles at each locus,wherein both alleles at substantially all of the loci consistessentially of the allele found at the same locus in corn variety LH249,the genome further comprising the genetic locus that confers the newtrait; and (f) harvesting the seed of the converted plant.
 20. Themethod of claim 19, wherein the genetic locus was stably inserted into acorn genome by genetic transformation.
 21. The method of claim 19,wherein the new trait is selected from the group consisting of herbicidetolerance; insect resistance; disease resistance; waxy starch; decreasedphytate content, modified fatty acid metabolism, modified carbohydratemetabolism; male sterility and restoration of male fertility.
 22. Aconverted plant of the corn variety LH249 produced by the method ofclaim
 19. 23. An F₁ hybrid seed produced by crossing the plant of claim2 with a second, distinct corn plant.
 24. An F₁ hybrid plant grown fromthe seed of claim 23.